The long-range goal of this research program is to understand the molecular basis of substrate specificity of glutathione transferases. These enzymes are a family of detoxification enzymes that are found in a wide range of species, including plants, insects and mammals. In humans, glutathione transferases play a role in the resistance towards carcinogens and the development of drug resistance of tumors to chemo-therapeutic drugs. An intriguing and functionally important property of these enzymes is their broad substrate specificity towards hydrophobic compounds. A single glutathione transferase is catalytically active on several different substrates. In addition, different glutathione transferases display different substrate specificities. The molecular mechanism of substrate specificity will be investigated by testing three, not necessarily exclusive, working hypotheses: 1. Broad substrate specificity may result from the existence of several functional hydrophobic binding sites contained within the active site regions. 2. Different glutathione transferases may utilize the free energy of substrate binding to alter the free energy of different positions along the reaction coordinate. The storage of free energy in different enzymes will be assessed by measuring the effect of ligand binding on amide exchange kinetics. 3. Protein dynamics may play a role in substrate binding and product release by gating access to the active site. Protein dynamics will be investigated by computer modeling, measurement of N-15 nuclear relaxation rates and by disulfide cross-linking. The largest difficulty in performing NMR experiments on these proteins is their size (55 KDa dimer). The high field TROSY spectra obtained at CMR will be invaluable in attaining the assignment of liganded and unliganded enzymes.